The major objective of this grant proposal is to further characterize the novel mitochondria-to-nucleus stress signaling discovered during the current grant period and to further establish its role in inducing tumor progression and invasive behavior. The stress signaling is initiated through changing Ca2+ fluxes and activation of Ca2+ responsive calcineurin and PKC. The nuclear target genes of stress signaling include those coding for mitochondrial proteins, and an array of extramitochondrial proteins such as ryanodine receptor Ca2+ channel 1, tumor specific markers cathepsin L, and TGFbeta3. Mitochondrial genetic stress (partial depletion of mtDNA) or metabolic stress (treatment with mitochondrial-specific inhibitors) induced invasive phenotypes in otherwise non-invasive C2C12 myocytes and human lung carcinoma A549 cells. The proposal is based on the hypothesis that mitochondrial stress induced activation of nuclear target genes involves a common co-activator protein and that stress signaling involving phenotypic changes presents an important risk factor in tumor progression and induction of invasive behavior. Following specific lines of experiments will be carried out to accomplish the goal: 1) The mitochondrial stress signaling in C2C12 rhabdomyoblasts and human lung carcinoma A549 cells activates calcineurin dependent NFAT and NFkB and PKC responsive Erg1, and Jun kinase activated ATF2 factors. Common mechanisms of stress induced transcriptional activation of nuclear target gene will be investigated using cathepsin L, RyR1, PEPCK (phosphoenolpyruvatecarboxykinase) and IRS1 (insulin receptor substrate 1) promoters as models. 2) Novel mechanism of mitochondrial stress-induced activation of NFkB through calcineurin-dependerit inactivation of lkBbeta will be further investigated using osteosarcoma TH1080 cells with mutated lkBalpha The distinctive nature of this signaling, and its target genes different from those regulated by the lkBalpha-dependent NFkB, will be investigated by transcriptional analysis of promoter constructs described above and also by microarray analysis. 3) The role of the mitochondrial compartment as the site of initiation of stress signaling will be further established using cybrid cells formed of cytoplast and nuclei from different sources. A conclusive evidence for the direct role of mitochondrial genetic or membrane systems can be provided by demonstrating that cytoplast from mtDNA-depleted cells when fused to nuclei from control cells induces phenotypic changes, invasive behavior, and resistance to apoptosis in the cybrid cells. The role of mitochondrial mutations in tumor invasion will be tested by creating cybrid cell lines containing nuclei from control or mtDNA-depleted A549 cells and cytoplasts from human tumor cells carrying homoplasmic mtDNA mutations.